The Role Of Autophagy/mitophagy In The Protection Effect Of Zinc On Hypoxia/reoxygenation Injury In Cardiac Cells

Objective To test if zinc protects cardiac cells from hypoxia/reoxygenation?H/R?injury by preventing mitochondrial oxidative stress through autophagy/mitophagy and to explore the mechanism underlying the induction of autophagy/mitophagy by zinc.Methods Cardiac H9c2 cells were subjected to hypoxia followed by reoxygenation.The mitochondrial morphological and functional changes,autophagosome,and autophagosomes enclosing mitochondria were detected by the transmission election microscopy?TEM?.The Western blotting analysis was used to determine the expression levels of LC3,TOM20,TIM23,COX4,Beclin1,PINK1,and the phosphorylation states of ERK,m TOR,AMPK,STAT3,and Bcl-2.The immunofluorescence imaging technique was used to determine the co-localization of LC3 and TOM20,MTG and PINK1.Fluorescence indicators including JC-1,DCFH-DA,and Mitosox Red were used to determine mitochondrial membrane potential???m?,reactive oxygen species?ROS?,and mitochondrial superoxide,respectively.Results1.Compared to the normal control,hypoxia/reoxygenation?H/R?markedly increased the cellular and mitochondrial ROS at reoxygenation,an effect that was prevented by the treatment of cells with ZnCl₂.This effect of zinc was reversed by the autophagic inhibitor 3-MA,suggesting that zinc prevents mitochondrial oxidative stress via autophagy.2.Under normoxic conditions,the LC3-II/I ratio was increased by ZnCl₂?1,5,and 10?M?in a dose-dependent manner with the peak at 5?M.ZnCl₂ also enhanced LC3-GFP fluorescence intensity.In addition,starvation?8 h?and rapamycin?20 ?M?increased LC3-II/I ratio and this was reversed by the selective zinc chelator TPEN?10 ?M?.The lysosomal blockade with Bafilomycin A1 further increased the effect of zinc on LC3-II/I ratio,indicating that zinc can promote the autophagic flow.All these data together indicate that exogenous and endogenous zinc can promote autophagy under physiological conditions.3.Under normoxic condtions,ZnCl₂ increased the co-localization of LC3?Green?and TOM20?Red?,and the autophagosomes enclosing mitochondria.Moreover,Western blotting analysis showed that the expression levels of the mitochondrial marker proteins including TOM20,TIM23,and COX4 were significantly decreased by ZnCl₂,implying that zinc can trigger mitophagy.4.In the setting of H/R,ZnCl₂ increased LC3-II/I ratio and Beclin1 expression but down-regulated TOM20,TIM23,and COX4.These effects of zinc were blocked by3-MA.These observations pointing to that zinc can also induce mitophagy in the setting of H/R.5.Compared to the H/R,ZnCl₂ increased ERK phosphorylation and the LC3-II/I ratio but reduced the levels of TOM20,TIM23,and COX4,an effect that was inhibited by the MEK inhibitor PD98059?2 ?M?,suggesting that ERK plays a role in the action of zinc on autophagy/mitophagy.6.Compared to the H/R,ZnCl₂ increased the Beclin1 expression and the LC3-II/I ratio,but decreased TOM20,TIM23 and COX4,which were inhibited by the transfection of cells with Beclin1 siRNA.In addition,PD98059 blocked the effect of ZnCl₂ on Beclin1 expression.These findings indicate Beclin1 is critical for the effect of zinc on mitophagy and ERK may serve as an upstream signal of Beclin1.7.Compared to the H/R,ZnCl₂ decreased TOM20,TIM23,and COX4,which was inhibited by PINK1 siRNA.ZnCl₂ increased the co-localization of Mitotracker Green and PINK1?red?during reoxygenation,which were inhibited by PD98059?2 ?M??P<0.05,respectively?,suggesting that PINK1 plays a role in the action of zinc on mitophagy at reoxygenation and ERK stabilizes PINK1 in mitochodnria.8.Compared to the H/R,zinc reduced the DCF?green?and Mito SOX Red?red?fluorescence intensities,an effect that was inhibited by Beclin1 and PINK1 siRNAs,implying that both Beclin1 and PINK1 are involved in the inhibitory effects of zinc on ROS generatioin.9.ZnCl₂ prevented H/R-induced collapse of ??m and increased cell viability,which was inhibited by Beclin1 and PINK1 siRNAs.Conclusions1.Both exogenous and endogenous zinc positively regulates autophagy.2.Zinc prevents mitochondrial ROS generation at reoxygenation by inducing autophagy/mitophagy. 3.ERK mediates the action of zinc on autophagy/mitophagy through Beclin1 and PINK1.